In spite of the inability of the gl60j;cryb double mutant to entrain to LD cycles, the behavior of such flies was still modified by the altered environmental conditions. This modulation of the activity level is interpreted as direct effects of light/radiant energy on locomotion that bypass the circadian system. Light-related energy often exerts such direct (or masking) effects on physiological parameters, including behavior. There are possibilities to distinguish real entrainment from masking: (1) after a phase shift of the LD cycle, the circadian rhythm often takes several transient cycles to reentrain
to the new LD regime, whereas masking follows the new light schedule immediately (in Drosophila, transients can be observed at very low light intensities or in photoreceptor mutants like cryb; (2) after transfer into
constant darkness, masking disappears immediately whereas an entrained rhythm starts to free run from the phase it had established in LD, and (3) masking is independent of a functional circadian clock -- for example,
it occurs in animals deprived of their clock, such as squirrel monkeys suffering from lesions of their suprachiasmatic nucleus arrhythmic per0 mutants of Drosophila (Helfrich-Forster, 2001).

In gl60j;cryb, prominent masking is observed at the highest light intensities employed (1000 lux). The sudden increase of the activity level after lights off and phase delay of the LD cycle did not show any transients after the 8 hr phase shift. Furthermore, this apparently forced activity disappears immediately after transfer into DD and independent of a functional clock, owing to its presence in these doubly mutant flies, which exhibit apparent rhythmicity in constant darkness. Moreover, no entrained cycling of clock protein levels was observed in these gl60j;cryb flies, demonstrating that the forced behavior is neither the consequence of molecular clock gene cyclings nor a nonphotic Zeitgeber that could influence the circadian clock (Helfrich-Forster, 2001).

In summary, the circadian blindness of flies expressing both glass and cryptochrome mutations is due to elimination of all photoreceptor cells that participate in entraining the circadian system. Similar complex light entrainment pathways may also exist in vertebrates. Interestingly, cryptochromes, certain opsins located in the retina, and standard photoreceptor cells
are candidates for participating in the circadian photoreception of mammals. Thus, rather than having an exclusive photopigment for entrainment of circadian rhythms, the situation in mammals could be similar to that in Drosophila: multiple photoreceptors share the workload involved in transmitting the principle environmental Zeitgeber to the circadian clock (Helfrich-Forster, 2001).

The extraretinal eyelet of Drosophila, the adult remnant of Bolwig's organ

Circadian rhythms can be entrained by light to follow the daily solar cycle. In
adult flies a pair of extraretinal eyelets expressing
immunoreactivity to Rhodopsin 6 each contains four photoreceptors located
beneath the posterior margin of the compound eye. Their axons project to the
region of the pacemaker center in the brain with a trajectory resembling that of
Bolwig's organ, the visual organ of the larva. A lacZ reporter line driven by an upstream fragment of the developmental gap gene Kruppel is a specific enhancer element for Bolwig's organ. Expression of immunoreactivity to the product of lacZ in Bolwig's organ persists through pupal metamorphosis and survives in the adult eyelet. It is thus demonstrated that the adult eyelet derives from the 12 photoreceptors of Bolwig's organ, which entrain circadian rhythmicity in the larva. Double labeling with anti-pigment-dispersing hormone shows that the terminals of Bolwig's nerve differentiate during metamorphosis in close temporal and spatial relationship to the ventral lateral neurons (LNv), which are essential to express circadian rhythmicity in the adult. Bolwig's organ also expresses immunoreactivity to Rhodopsin 6, which thus continues to be expressed in the adult eyelet. Action spectra of entrainment were compared in different fly strains: in flies lacking compound eyes but retaining the adult eyelet (so1), lacking both compound eyes and the adult eyelet (so1;gl60j), and retaining the adult eyelet but lacking compound eyes as well as Cryptochrome (so1;cryb). Responses to phase shifts suggest that, in the absence of compound eyes, the eyelet together with Cryptochrome mainly mediates phase delays. Thus a functional role in circadian entrainment first found in Bolwig's organ in the larva is retained in the eyelet, the adult remnant of Bolwig's organ, even in the face of metamorphic restructuring (Helfrich-Forster, 2002).

Circadian rhythms are orchestrated by a master clock that emerges from a network of circadian pacemaker neurons. The master clock is synchronized to external light/dark cycles through photoentrainment, but the circuit mechanisms underlying visual photoentrainment remain largely unknown. This study reports that Drosophila has eye-mediated photoentrainment via a parallel pacemaker neuron organization. Patch-clamp recordings of central circadian pacemaker neurons reveal that light excites most of them independently of one another. Light-responding pacemaker neurons were shown to send their dendrites to a neuropil called accessary medulla (aMe), where they make monosynaptic connections with Hofbauer-Buchner eyelet photoreceptors and interneurons that transmit compound-eye signals. Laser ablation of aMe and eye removal both abolish light responses of circadian pacemaker neurons, revealing aMe as a hub to channel eye inputs to central circadian clock. Taken together, this study demonstrates that the central clock receives eye inputs via hub-organized parallel circuits in Drosophila (Li, 2018).

tll controls a switch between optic lobe
and Bolwig's organ cell fate.
Loss of zygotic tll activity results in an absence
of most of the protocerebrum of the brain
(Younossi-Hartenstein, 1997). In addition, the visual
system of the late tll embryo shows a dramatic phenotype,
namely the transformation of optic lobe into Bolwig's organ. In wild-type embryos, the neuronal marker 22C10 (see Futsch)
labels 12 neurons and their axons that project towards the optic
lobe. In tll mutant embryos, the number of cells in the Bolwig's
organ is dramatically increased (by a factor of 2-3),
while the optic lobe, marked by anti-Crumbs or a
PlacZ insertion in so, is absent.
Use of antibodies to FasII and Crb, which label the apical
surface of the optic lobe, and not that of the Bolwig's organ,
allowed an analysis of how the phenotype unfolds in the tll
mutant embryo. Abnormalities first become apparent during
stage 11, when FasII expression increases strongly in the domain
of the anterior lip of the optic lobe placode, a region that
normally ceases to express FasII. In spite of this
abnormal expression, the optic lobe placode appears to
invaginate normally. As a results, in stage 13 tll mutant embryos,
a Crb-positive vesicle can be seen subjacent to the head
epidermis. During
stage 14, all cells of this aberrant vesicle activate expression of
the neuronal marker 22C10 and lose Crb expression, revealing that these cells are Bolwig's organ cells.
Overexpression of tll under the control of a heat-shock
promoter has an effect opposite that seen in the absence of
tll activity. An
additional consequence of tll overexpression is that the optic
lobes become located more dorsally and fused in the dorsal
midline. This 'cyclops' phenotype most likely arises
because the dorsomedial cells, which normally
die or form part of the head epidermis, now express optic lobe
markers and become an integral part of the optic lobe.
The
results of both loss and gain of tll expression are consistent
with the interpretation that tll is required to drive cells of the
anlage, which would otherwise become photoreceptor neurons
of Bolwig's organ, to develop as optic lobe cells (Daniel, 1999).

>atonal is expressed in and required for the development of Bolwig's organ. >ato is expressed in the head in
several small cell clusters, one of which is a group of three to
four cells that is part of the Bolwig's organ primordium.
Expression of >ato in this domain begins during stage 11 and continues until stage 12. Initially, a group of
6-8 cells faintly expresses >ato. By stage 12, their number has
decreased to 3 cells. During this period, >ato-expressing cells
can be seen as a small group of cells within the dome-shaped
Bolwig's organ primordium. Loss of >ato function results in the
absence of Bolwig's organ. Thus, similar to what has
been demonstrated for the compound eye, even though only a
small subset of photoreceptors actually expresses >ato, lack of
>ato function results in absence of all photoreceptors.
Since Bolwig's organ is enlarged in a tll mutant background,
it was asked whether tll inhibits >ato expression. The number and pattern of >ato-positive cells in tll mutants is found to be
normal. These results suggest that tll functions in
parallel with, or downstream of >ato in the development of the
Bolwig's organ/optic lobe primordium (Daniel, 1999).

Epidermal growth factor receptor is activated in midline
regions of the head neurectoderm, in particular in the anlage
of the visual system. Moreover,
increased and/or ectopic activation of Egfr results in a
'cyclops' phenotype very similar to what has been described for
ectopic tll expression. Egfr signaling has been shown to be
required in both chordotonal organs and compound eye
for the inductive signaling triggered by >ato expression. Two questions raised by these observations have been investigated:
(1) is Egfr signaling required for tll expression in the optic
lobe and
(2) is Egfr signaling involved in the recruitment
of the secondary (non->atonal-expressing) Bolwig's
organ cells? The answer to both these questions is no. tll expression is unaltered when levels of Egfr signaling are manipulated, suggesting that Egfr signaling is not required for tll expression. To investigate the second question, a test was performed for
the presence of Egfr-relevant mRNAs or proteins:
Rhomboid mRNA, which would be expected to be present
only in the signaling cells, and phosphorylated
MAPK, Pointed and Argos mRNAs, which would be
expected to be expressed in all cells receiving an
Egfr-mediated signal. In stage 12 embryos, rho is
expressed only in the small group of Bolwig's organ
founder cells (the same cells expressing >ato).
In contrast, activated (phosphorylated) MAPK is
present in a larger cluster of cells including the entire
Bolwig's organ and part of the adjacent optic lobe. Consistent with this, pnt and aos, both
known to be switched on in cells receiving the Spi
signal, are expressed at the same stage throughout the
entire Bolwig's organ primordium.
These gene expression and MAPK activation
patterns are consistent with the idea that the Spi signal
is activated by rho in the Bolwig's organ founders and
passed to the neighboring secondary Bolwig's organ
cells where it activates the Egfr signaling cascade.
Supporting this view, only 3-4 photoreceptor neurons
are found in the Bolwig's organ of embryos lacking
rho or spi; furthermore, the size of the
posterior lip of the optic lobe is reduced in such
embryos. The fact that absence of
secondary Bolwig's organ cells in rho or spi mutant
embryos can be rescued by blocking cell death in the
background of a deficiency that takes out the reaper
complex of genes indicates that the Spi signal is not necessary
for the specification (recruitment) of secondary Bolwig's organ
cells, but rather, for their maintenance (Daniel, 1999).

While the maternal patterning systems that regulate
tll during its blastoderm expression have been
determined, the genes required
to turn on tll at a later stage in the visual system are
not known. Candidates are the 'early eye genes', so,
eya and ey, which encode transcription factors
expressed in the embryonic visual system and in the
larval eye disc in front of the morphogenetic furrow. The expression of these genes was analyzed in the visual system
anlage, and tll expression was examined in embryos mutant
for each of these genes. tll expression
in the optic lobe does not depend on any of these three genes.
It was also concluded that ey and so, which have been shown
to interact with each other during eye disc determination, must act independently in embryonic
visual system development, since they are expressed in those
primordia in non-overlapping patterns (Daniel, 1999).

Although so is expressed initially in the entire visual system
anlage, during later stages its expression
becomes increasingly restricted to subsets of visual system
progenitors. Thus, during stage 11, when a morphologically
distinct optic lobe placode first becomes visible, the domain of
so expression retreats to the posterior lip of this placode; slightly
later its expression is limited to only the Bolwig's organ, where
it is maintained until stage 13. eya expression is
initiated during the late blastoderm stage in a trapezoidal field
in the dorsomedial head region that includes the visual system
anlage, as well as progenitors of the medial brain. Beginning during gastrulation (stage 6/7), the eya
domain becomes divided into an anterior stripe and a narrow
posterior stripe immediately anterior to the cephalic furrow that
widens laterally; this posterior domain will become part of the
posterior lip of the optic lobe, including Bolwig's organ. eya expression continues in the optic lobe until stage 12 and
in Bolwig's organ until stage 13.
Embryos that lack either so or eya exhibit defects in the
portions of the visual system where these genes are expressed.
In both mutants, development proceeds normally until stage
11, when the posterior lip of the optic lobe (olpl) would
normally start to invaginate. In eya and so embryos,
invagination of the optic lobe placode does not take place and
differentiation markers characteristic of the lobe are not expressed.
In conclusion, so and eya, although
expressed coincidental with tll, are not required for its
activation. ey plays no role in the embryonic visual system (Daniel, 1999).

Since tll is a nuclear receptor transcription factor, it must
function to block the effect of signaling from the founder cells
(which is mediated at least in part by Egfr)
at the transcriptional level. In the posterior of the blastoderm
stage embryo, tll has been shown to function directly as both
a repressor (of Kruppel, knirps and Ubx) and as an activator
(of hunchback). Additional activator effects
of tll (not yet demonstrated to be direct) have been shown for
brachyenteron at the posterior of the blastoderm embryo, and
for the proneural gene lethal of scute in the brain. Thus, in the optic
lobe, tll could repress genes that would in its absence be
activated by Egfr signaling, and/or activate genes that would
block receipt, or execution, of the signal (Daniel, 1999 and references).

Light-induced structural and functional plasticity in Drosophila larval visual system

How to build and maintain a reliable yet flexible circuit is a fundamental question in neurobiology. The nervous system has the capacity for undergoing modifications to adapt to the changing environment while maintaining its stability through compensatory mechanisms, such as synaptic homeostasis. This study describes findings in the Drosophila larval visual system, where the variation of sensory inputs induces substantial structural plasticity in dendritic arbors of the postsynaptic neuron and concomitant changes to its physiological output. Furthermore, a genetic analysis has identified the cyclic adenosine monophosphate (cAMP) pathway and a previously uncharacterized cell surface molecule as critical components in regulating experience-dependent modification of the postsynaptic dendrite morphology in Drosophila (Yuan, 2011).

Proper functions of neuronal circuits rely on their fidelity, as well as plasticity, in responding to experience or changing environment, including the Hebbian form of plasticity, such as long-term potentiation, and the homeostatic plasticity important for stabilizing the circuit. Activity-dependent modification of neuronal circuits helps to establish and refine the nervous system and provides the cellular correlate for cognitive functions, such as learning and memory. Multiple studies have examined synaptic strength regulation by neuronal activity, whereas to what extent and how the dendritic morphology may be modified by neuronal activity remain open questions (Yuan, 2011).

The model organism Drosophila melanogaster has facilitated genetic studies of nervous system development and remodeling. Notwithstanding the relatively stereotyped circuitry, flies exhibit experience-induced alterations in neuronal structures and behaviors such as learning and memory). In a study of experience-dependent modifications of the Drosophila larval CNS, it has been found that different light exposures dramatically altered dendritic arbors of ventral lateral neurons [LN(v)s], which are postsynaptic to the photoreceptors. Unlike the visual activity-induced dendrite growth in Xenopus optic tectum, extending the light exposure of Drosophila larvae reduced the LN(v)s' dendrite length and functional output, a homeostatic plasticity for compensatory adaptation to alterations in sensory inputs. It was further shown that the cyclic adenosine monophosphate (cAMP) pathway and an immunoglobulin domain-containing cell surface protein, CG3624, are critical for this sensory experience-induced structural plasticity in Drosophila CNS (Yuan, 2011).

In Drosophila larvae, Bolwig's organ (BO) senses light, and its likely postsynaptic targets are LN(v)s. As the major circadian pacemaker, LN(v)s are important for the entrainment to environmental light-dark cycles and larval light avoidance behavior. In the larval brain, Bolwig's nerve (BN), the axonal projection from BO, terminates in an area overlapping the dendritic field of LN(v)s. Using the FRT-FLP system [in which DNA sequences flanked by flippase recognition targets (FRT) are snipped out by flippase (FLP)] along with three-dimensional (3D) tracing, the dendritic arbor of individual LN(v) neurons were labeled and analyzed. Then potential synaptic connections were demonstrated between BN and LN(v)s using the GRASP [green fluorescent protein (GFP) reconstitution across synaptic partners] technique to drive expression of one-half of the split GFP in the BN by means of Gal4/UAS and expression of the other half of the split GFP in LN(v)s via LexA/LexAop. The proximity of putative synaptic connections between BN and LN(v)s' dendrites reconstituted GFP fluorescence for photoreceptors expressing either rhodopsin 5 (Rh5) or rhodopsin 6 (Rh6) in BO, which suggested that both groups of photoreceptors may have synaptic connections with LN(v)s (Yuan, 2011).

To test whether LN(v)s can be activated by BN inputs through light stimulation, calcium imaging was performed using GCaMP3 transgenic flies with the larval brain-eye preparation, which included BO, BN, developing eye disks, the larval brain, and ventral nerve cord. Because BO senses blue and green light, the confocal laser at 488 nm (blue) and 543 nm (green) were used to stimulate these larval photoreceptors. LN(v)s' axon terminals displayed a relatively stable baseline of GCaMP3 fluorescence and, upon light stimulation, yielded large calcium responses, which increased with the greater intensity and longer duration of the light pulses (Yuan, 2011).

Recent studies suggest that Cryptochrome (CRY) in adult large LN(v)s senses light and elicits neuronal firing. In larvae, however, severing BN abolished light-induced calcium responses in LN(v)s. The loss-of-function mutation of NorpA (no-receptor-potential A), encoding a phospholipase C crucial for phototransduction, also eliminated these calcium responses, which indicated that light-elicited responses in LN(v)s are generated via phototransduction in larval photoreceptors rather than as a direct response to light by LN(v)s (Yuan, 2011).

In animals with BO genetically ablated, the dendritic field of LN(v) is absent. To test whether BO is required for LN(v)s' dendrite maintenance, the expression of cell death genes rpr and hid was induced in BO after synapse formation, and the LN(v) dendrite length was also found to be greatly reduced. Whereas physical contacts with BN or growth-promoting factors released from presynaptic axons could be important for LN(v)s' dendrite maintenance, it is also possible that synaptic activity from BN promotes LN(v) dendrite growth, as suggested by previous studies. To explore the latter scenario, newly hatched larvae were provided with different visual experiences through various light regimes—including the standard 12 hours of light and 12 hours of dark daily cycle (LD); constant darkness (DD) for sensory deprivation; constant light (LL) for enhanced light input; 16-hour light and 8-hour dark cycle, mimicking a long day; and 8-hour light and 16-hour dark cycle, mimicking a short day. The dendrite morphology of LN(v)s of late third instar larvae was examined. Whereas different light exposure had no detectable effects on larval developmental timing, increasing light exposure reduced the total dendrite length of individual LN(v) neurons, with the longest dendrite in constant darkness and the shortest dendrite length in constant light condition. Thus, not only is the LN(v) dendrite dependent on the presence of presynaptic nerve fibers, its length is modified by the sensory experience in a compensatory fashion, whereby an increase in sensory inputs causes a reduction in the dendrite length and vice versa (Yuan, 2011).

Whereas adult LN(v)s alter their axon terminal structures in a circadian cycle-controlled fashion, no difference was found in dendrite morphology of LN(v)s from larvae collected at four different time points around the clock, which indicated that circadian regulation is not involved in the light-induced modification of LN(v) dendrites. Under regular light-dark conditions, LN(v) dendrites expanded as the larval brain size increased from the second to the third instar stage. However, the dendrite length of the LL group increased at a significantly slower rate than the DD group. It thus appears that light exposure retards the growth of LN(v) dendrites throughout the larval development (Yuan, 2011).

To test the contribution of different light-sensing pathways, loss-of-function mutations of Cry (cry01) or NorpA (norpA36) and of double mutants lacking both Rh5 and Rh6 (rh52;rh61) were examined. Although wild-type and cry01 larvae displayed differences in their dendrite length when exposed to constant darkness versus constant light, such light-induced changes were absent in the rh52;rh61 double mutant and the norpA36 mutant. Thus, similar to the calcium response to light, light-induced modification of LN(v) dendritic structure requires visual transduction mediated by rhodopsin and NorpA in BO but not Cry function in LN(v)s (Yuan, 2011).

To manipulate the level of synaptic activity, the BO excitability was weither increased by expressing the heat-activated Drosophila transient-receptor-potential A1 (dTrpA) channel, or transmitter release from BN was reduced through a temperature-sensitive form of the dominant-negative dynamin, Shibirets (Shits). These manipulations eliminated light-induced modification of LN(v) dendrites at 29°C. Reducing BO activity by means of Shits caused dendrite expansion, as if the animal detected no light, whereas increasing BO activity by means of the dTrpA channel resulted in reduction of LN(v) dendrites, a process reminiscent of constant light exposure (Yuan, 2011).

Whether intrinsic LN(v) neuronal activity drives modification of its dendrite morphology was further tested by expression of either the sodium channel NaChBac to increase excitability or the potassium channel Kir2.1 to reduce excitability. LN(v)s expressing Kir2.1 showed reduced or no calcium responses upon light stimulation. In contrast, LN(v)s expressing NaChBac displayed numerous peaks in GCaMP3 signals in the presence or absence of light stimulation, indicative of elevated spontaneous activities. Upon examining LN(v) dendrites, it was found that neuronal excitability of the LN(v) was inversely proportional to its dendrite length (Yuan, 2011).

These results obtained using genetic approaches agreed with findings in experiments with different environmental light conditions. They suggested that LN(v)'s dendritic structures are modified according to its neuronal activity, which varies with light-induced synaptic inputs (Yuan, 2011).

To test whether synaptic contacts of BN on LN(v)s are modified by light, synapses formed by BN with EGFP (enhanced green fluorescent protein)-tagged Cacophony (Cac-EGFP) were marked, because Cacophony is a calcium channel localized at presynaptic terminals and its distribution correlates with the number of synapses. Close association was found of Cac-EGFP-expressing structures with LN(v)s' dendritic arbors. Compared with regular light-dark conditions, constant darkness increased, whereas constant light reduced, the total intensity of Cac-EGFP, which suggested that light modified not only dendritic arbors of LN(v)s but also the number of synaptic contacts impinging on LN(v) dendrites (Yuan, 2011).

Next, using calcium imaging, whether there are light-induced functional modifications of LN(v)s was examined. Increased light exposure caused LN(v)s to be less responsive. Conversely, sensory deprivation in constant darkness increased LN(v)s' sensitivity to light. Thus, in contrast to stable synaptic responses observed in synaptic homeostasis, light-induced responses of central neurons postsynaptic to photoreceptors in the Drosophila larval visual circuit have a dynamic range, modifiable by sensory experiences and positively correlated to the dendrite length (Yuan, 2011).

In dunce1, a loss-of-function mutant of the fly homolog of 3'5'-cyclic nucleotide phosphodiesterase, the LN(v)s' dendrite length was comparable among LD, LL, and DD groups. Reducing dunce gene expression specifically in LN(v)s through RNA interference (dncIR) resulted in a similar indifference of LN(v)s' dendrite size to the light exposure, which implicated a cell-autonomous action of dunce in LN(v) neurons (Yuan, 2011).

To explore the possibility that the elevated cAMP level caused by the dunce mutation interfered with dendrite plasticity, tests were performed for the involvement of downstream components of the cAMP pathway, including the catalytic subunit of protein kinase A (PKAmc), which up-regulates cAMP signaling, and a dominant-negative form of the cAMP response element-binding protein (CREBdn), which inhibits cAMP-induced transcription activation. Expression of either transgene specifically in LN(v)s obliterated their ability to adjust dendrite length under different light-dark conditions. Calcium imaging further revealed that the expression of PKAmc or CREBdn eliminated changes of LN(v)s' light responses produced by different light-dark conditions. Thus, the cAMP pathway regulates both structural and functional plasticity of LN(v)s (Yuan, 2011).

The screen for mutants with defective LN(v) dendritic plasticity also identified babos-1, a mutant with a P-element insertion near the transcriptional start site of CG3624, a previously uncharacterized immunoglobulin domain-containing cell surface protein. The LN(v) dendrite length of babos-1 mutant larvae was comparable to controls in LD and LL but has no compensatory increase in DD. Similar phenotypes were found in larvae expressing an RNAi transgene targeting CG3624 in LN(v)s. Moreover, flies carrying a hypomorphic allele of CG3624, CG3624[KG05061], also showed defective light-induced dendritic plasticity, which was fully rescued by expressing the UAS-CG3624 transgene specifically in LN(v)s. Thus, the function of this immunoglobulin domain-containing protein in LN(v)s is important for the dendrite expansion in constant darkness (Yuan, 2011).

Bioinformatic analyses suggest that CG3624 is a cell surface protein containing an N-terminal signal peptide, extracellular immunoglobulin domains followed by a transmembrane helix, and a short C-terminal cytoplasmic tail. CG3624 is widely expressed in the nervous system throughout development. Its specific requirement for the adjustment of LN(v)s' dendrite length in constant darkness suggests that elevation or reduction of sensory inputs likely invokes separate mechanisms for compensatory modifications of central neuronal dendrites (Yuan, 2011).

A functioning nervous system must have the capacity for adaptive modifications while maintaining circuit stability. This study of the Drosophila larval visual circuit reveals large-scale, bidirectional structural adaptations in dendritic arbors invoked by different sensory exposure. Whereas the circuit remains functional with modified outputs, this type of homeostatic compensation may modify larval light sensitivity according to its exposure during development and could facilitate adaption of fly larvae toward altered light conditions, such as seasonal changes. The observations also suggest shared molecular machinery between homeostasis and the Hebbian plasticity with respect to the cAMP pathway and indicate the feasibility of genetic studies of experience-dependent neuronal plasticity in Drosophila (Yuan, 2011).

In addition to the compound eyes, most insects possess a set of three dorsal ocelli that develop at the vertices of a triangular cuticle patch, forming the ocellar complex. The wingless and hedgehog signaling pathways, together with the transcription factor encoded by orthodenticle, are known to play major roles in the specification and patterning of the ocellar complex. Specifically, hedgehog is responsible for the choice between ocellus and cuticle fates within the ocellar complex primordium. However, the interaction between signals and transcription factors known to date do not fully explain how this choice is controlled. This study shows that this binary choice depends on dynamic changes in the domains of hedgehog signaling. In this dynamics, the restricted expression of engrailed, a hedgehog-signaling target, is key because it defines a domain within the complex where hh transcription is maintained while the pathway activity is blocked. The Drosophila Six3, Optix, is expressed in and required for the development of the anterior ocellus specifically. Optix would not act as an ocellar selector, but rather as a patterning gene, limiting the en expression domain. These results indicate that, despite their genetic and structural similarity, anterior and posterior ocelli are under different genetic control (Dominguez-Cejudo, 2015).

The dorsal adult head of Drosophila derives from the dorsal-anterior region of the eye-antennal imaginal disc. In addition, this disc gives rise to the remaining head capsule, the eyes, the antennae and the maxillary palps. The dorsal head is patterned by the dynamic interplay between orthodenticle [otd; also known as ocelliless (oc)], which encodes an Otx family transcription factor, and the wingless (wg; the fly Wnt1 homolog) and hedgehog (hh) signaling pathways . The result of this patterning is the allocation of the dorsal head, which lies in between the eyes, into three territories (from lateral to medial): orbital cuticle, frons and ocellar complex (OCx). The OCx comprises three small and structurally simple eyes termed the ocelli that are located at the vertices of a triangular patch of cuticle, the so-called interocellar cuticle, which also harbors a set of stereotypical bristles. Ocelli are widespread in insects, where they play a number of roles, including flight stabilization and as movement detectors triggering the escape response (Dominguez-Cejudo, 2015).

Work in past years has aimed at defining the functional relationships between wg, hh and otd during the process of dorsal head patterning in the disc. This work has expanded understanding of the general mechanisms by which the conserved Wnt and Hh signaling pathways interact and of the development and evolution of the eyes and dorsal head of arthropods, and has helped in establishing parallels between head patterning across phyla. For instance, members of both the Wnt and Otx gene families are involved in anterior head/neural tube patterning in both invertebrates and vertebrates (Dominguez-Cejudo, 2015).

The development of the OCx is a typical example of regional specification, in which the OCx progenitor field is further subdivided to give rise to the three ocelli and the interocellar cuticle. One of the earliest steps during the development of the Drosophila head is the initiation of otd expression by wg. In late embryos, all cells of the eye-antennal disc primordium, which can be marked by the expression of eyeless (ey), express Otd. During larval development Otd expression progressively disappears from the disc, and is only maintained in its dorsal anterior region, where wg is expressed. Otd in turn is required to activate hh transcription. This results, in the early third instar (L3) disc, in the coexpression of wg, hh and otd in the prospective OCx region. However, wg transcription is next repressed in the prospective OCx to allow the development of the ocelli and the interocellar cuticle and bristles; otherwise, these structures fail to develop and are replaced by frons, a more lateral type of cuticle (Dominguez-Cejudo, 2015).

During mid and late L3, the OCx region becomes further subdivided into three domains: the central domain transcribes hh and becomes the interocellar cuticle (IOC) region, while two adjacent domains express eyes absent (eya) and sine oculis (so), which encodes a Six1/2 transcription factor, and will become the anterior (a) and posterior (p) ocelli (OC) (Blanco, 2010; Brockmann, 2011). The mechanism by which this aOC-IOC-pOC pattern is controlled by hh has recently been investigated (Aguilar-Hidalgo, 2013) and relies on the differential activation by the Hh signaling pathway of two Hh target genes: eya and the homeobox transcription factor engrailed (en). Hh first activates eya throughout the OCx region; then, en is turned on in a more restricted domain, which results in the attenuation of the Hh signaling pathway and the concomitant loss of eya from these cells. Thus, the central region, expressing en and devoid of eya, becomes the IOC, whereas the remaining flanking eya-expressing domains become the retina-producing OC (Dominguez-Cejudo, 2015).

However, a central question that remains to be answered for a comprehensive understanding of ocellar specification is how the changes in hh signaling domains are regulated during development, as this signaling morphogen plays a major role in controlling the specification and patterning of the OCx structures. This study has followed the regulatory steps that lead from the onset of hh expression to the establishment of its final expression domain, and defines how these steps are interconnected in a gene regulatory network. Two transcriptional repressors, encoded by en and the Drosophila Six3/6 gene Optix, are key players in this network (Dominguez-Cejudo, 2015).

The relative simplicity of the OCx makes it an ideal system with which to study in detail the mechanisms involved in the specification and patterning of a visual structure. Previous work had described the functional relationships between wg, hh and otd during the specification of the dorsal head, the region where the OCx forms. The outcome of these interactions, a wg-cleared OCx region, allows the subsequent specification of the ocellar structures, namely the three ocelli (an eya/so-dependent structure) and the intervening interocellar cuticle (an en-dependent structure), by the Hh signaling pathway. However, it is not clear how this aOC-IOC-pOC pattern is generated. More specifically, since this pattern depends on hh, the question is to understand how hh controls alternative fate decisions in this region. This work has shown that the hh signaling domain changes during this process and that this change is essential for proper ocellar development. This dynamics depends on the establishment of a feedback loop with the hh signaling pathway target en, which, in turn, is restricted in its expression domain by the action of the Drosophila Six3/6 homolog Optix (Dominguez-Cejudo, 2015).

During the first half of L3 two Hh-related events occur. First, wg transcription clears from the prospective ocellar region. This is mediated by high Otd levels, which are achieved through the activation of an otd autoregulatory enhancer [oc7 (Blanco, 2009)] by Hh signaling. Second, and parallel to the wg clearing, low levels of eya expression are induced throughout the whole OCx (Aguilar-Hidalgo, 2013). During the second half of L3, though, the initially uniform expression domain of hh fades away to become restricted to its central region, associated with the activation of en. This central domain becomes the non-retinal interocellar cuticle (IOC), where en represses the transduction of the hh signal. This change in hh expression pattern, which defines the aOC-IOC-pOC domain organization in the disc and the structure of the adult OCx, occurs through transcriptional changes. In particular, the maintenance of hh in the central domain depends on en. Therefore, after en expression is turned on by hh signaling (Aguilar-Hidalgo, 2013), en feeds back positively on hh transcription to maintain high hh expression levels. In the prospective IOC, the expression of en represses hh signaling transduction and the initial expression of eya is lost. In the adjacent regions, though, eya expression is maintained at high levels through an autoregulatory loop that involves so (Brockmann, 2011). In addition, a potential non-autonomous contribution of Hh, produced at the IOC region, cannot be excluded. The en-to-hh maintenance function that is described in this study in the OCx resembles the well-established role of en as a hh transcriptional activator in other contexts, such as the embryo segmental stripes and the posterior compartment of the wing disc, and could constitute a regulatory module that is deployed in several developmental contexts, such as the developing head (Dominguez-Cejudo, 2015).

It was further demonstrated that the peripheral reduction of the hh domain is due to transcriptional regulation rather than cellular rearrangements. This raises the question of how the reduction of hh transcription outside the IOC region occurs. The most likely possibility is that a hh activator is lost as the development of the OCx region progresses through L3. It is noted that hyperactivation of the wg canonical pathway in the OCx region results in an expansion of the hh-Z domain. If wg were required to activate hh, the indirect negative-feedback loop that results in the wg clearing from the OCx region would also result in the loss of its activating action on hh and the loss of hh transcription. This would be prevented only in places where en was expressed. This hypothesis (wg being required for hh expression in the OCx) is supported by the fact that, in the embryonic head, wg activates hh expression. Nevertheless, it seems contradictory to previous reports in which, using a temperature-sensitive wg allelic combination, the reduction of wg signaling activity resulted in an enlargement of the OC and the IOC. However, this result could be reconciled with wg acting as a hh activator. Early during L3, wg would activate hh transcription while simultaneously preventing the expression of en and eya, two targets of hh (Blanco, 2009). Later in L3, high levels of Otd, produced after the activation of the oc7 enhancer, result in the transcriptional repression of wg in the prospective OCx region. This repressive step leaves the wg expression domain restricted to more lateral regions, where the frons and the orbital cuticle will be specified. Removing wg function during this late period, would allow the activation of Hh targets eya and en in a broader domain due to the non-autonomous action of secreted Hh. Since en maintains hh transcription at high levels, late removal of wg should result in an enlarged hh expression domain and an increase in the overall size of the OCx, as observed. It is also important to mention that previous work has shown that the Iroquois Complex (Iro-C) genes araucan and caupolican participate in the restriction of the OCx to the medial region (Yorimitsu, 2011; Dominguez-Cejudo, 2015).

Key to the establishment of OCx patterning is where en becomes expressed. In contrast to hh transcription, which changes over time, that of en is stable once initiated in the prospective IOC. Optix is expressed in a dorsal anterior strip in the eye disc, contained within the otd domain, that abuts posteriorly the en domain. The results suggest that Optix is partially responsible for setting up this anterior border of en. Since en, by acting as a Hh pathway repressor, prevents eya transcription, such an expansion is the most likely cause of the effects on the aOC. The definition of a clear-cut Optix/en border might be further refined by mutually repressive interactions, as indicated by two results: overexpressing Optix in the IOC results in the downregulation of en and, reciprocally, the overexpression of en in the prospective aOC represses Optix. Therefore, as en is initially activated by hh, the anterior limit of the en domain may result from the integration of activator and repressor inputs provided by hh signaling and Optix, respectively, a limit that might be further refined by reciprocal repression of Optix by en. It is hypothesized that a similar mechanism sets the posterior border of the en expression domain to allow for an en-free, hh-receiving domain that becomes specified as the pOC. Potential candidates for the posterior anti-repressor are the Sp genes buttonhead (btd) and Sp1, and hunchback (hb). These genes are expressed in the preoptic region of the embryonic head of all arthropods. However, neither Sp1 nor btd is expressed in the prospective OCx region of the eye disc. hb expression was checked using an anti-Hb antibody and no expression was found in L3 eye-antennal discs. Therefore, the nature of the posterior regulator(s) involved remains to be determined (Dominguez-Cejudo, 2015).

The results indicate that, despite their structural similarity and shared requirement of otd, hh signaling and eya activation, the patterning of the aOC and pOC are under different genetic control. This might be expected, as only the anterior ocellar patches fuse during metamorphosis to form the adult aOC. In fact, evidence for this difference in genetic control had previously been obtained in population selection experiments in Drosophila suboscura. In that study, the authors used an ocelliless (oc) mutant population that showed loss of OCx structures, including the aOC and pOC, with variable penetrance. Through breeding, they were able to establish independent sublines in which the aOC, but not the pOC (or vice versa), were preferentially lost, even when these flies were still carrying the otd mutation. In light of these results, it was proposed that, on top of a common precursor for OC and bristle (i.e. cuticle) determined by otd, an additional 'system' would control the amount of ocellar or cuticle precursors, and this system would differ along the anterior-posterior axis. In this context, Optix would not instruct an ocellar fate but rather control the amount of anterior ocellus precursor cells within the OCx primordium, thus acting as a pre-patterning gene (Dominguez-Cejudo, 2015).

The expression pattern and function of six3/Optix have been studied in Drosophila embryos. In both insects, six3/Optix expression is restricted to the head region, and includes the clypeolabrum and maxillary segment in Drosophila and the labral and middle head regions in Tribolium. Accordingly, six3/Optix mutant Drosophila larvae show reduced or absent labral-derived head skeletal elements, such as the labral organ and the maxillary segment-derived mouth hooks in Drosophila and loss of the labrum and anterior vertex bristle in Tribolium. This study has shown that in Drosophila the expression domain of Optix in eye discs, which runs along the anteriormost medial disc region, maps to the anterior medial dorsal head, where it is required. In addition, OptixNP2631>OptixRNAi adults show defects in the clypeal skeleton, recapitulating the defects seen in larvae. Therefore, all these results point to six3/Optix as a medial head-patterning gene also during eye-antennal disc development. However, it was not been able to detect Optix expression in the embryonic primordium of the eye-antennal disc, marked with the disc primordium marker ey-Z and using either an anti-Optix antiserum or the OptixNP2631-GAL4 line. The expression of all available regulatory constructs associated with the Optix locus generated by the Janelia Project was studied. Neither of the lines expressed in the eye disc showed overlapping expression with the embryonic eye primordium. Therefore, and provided that a low number of Optix-expressing cells in the eye-antennal disc primordium was not missed, the expression of Optix in the head primordium is likely to be initiated during larval life (Dominguez-Cejudo, 2015).

The mechanisms that initiate Optix expression in the most anterior region of the dorsal head need to be investigated further. The results also raise the question of whether en, otd and hh might be similarly engaged in adult head patterning in other insects (Dominguez-Cejudo, 2015).

A sensitivity of the circadian clock to light/dark cycles ensures that biological rhythms maintain optimal phase relationships with the external day. In animals, the circadian clock neuron network (CCNN) driving sleep/activity rhythms receives light input from multiple photoreceptors, but how these photoreceptors modulate CCNN components is not well understood. This study shows that the Hofbauer-Buchner eyelets, located between the retina and the medulla in the fly optic lobes, differentially modulate two classes of ventral lateral neurons (LNvs) within the Drosophila CCNN. The eyelets antagonize Cryptochrome (CRY)- and compound-eye-based photoreception in the large LNvs while synergizing CRY-mediated photoreception in the small LNvs. Furthermore, it was shown that the large LNvs interact with subsets of "evening cells" to adjust the timing of the evening peak of activity in a day length-dependent manner. This work identifies a peptidergic connection between the large LNvs and a group of evening cells that is critical for the seasonal adjustment of circadian rhythms (Schlichting, 2016).

Circadian clocks create an endogenous sense of time that is used to produce daily rhythms in physiology and behavior. A defining characteristic of a circadian clock is a modest deviation of its endogenous period from the 24.0 h period of daily environmental change. For example, the average human clock has an endogenous period of 24 h and 11 min. Thus, to maintain a consistent phase relationship with the environment, the human clock must be sped up by 11 min every day. A sensitivity of the circadian clock to environmental time cues (zeitgebers) ensures that circadian clocks are adjusted daily to match the period of environmental change. This process, called entrainment, is fundamental to the proper daily timing of behavior and physiology. For most organisms, daily light/dark (LD) cycles are the most salient zeitgeber (Schlichting, 2016).

Although most tissues express molecular circadian clocks in animals, the clock is required in small islands of neural tissue for the presence of sleep/activity rhythms and many other daily rhythms in physiology. Within these islands, a circadian clock neuron network (CCNN) functions as the master circadian clock. Subsets of neurons within the CCNN receive resetting signals from photoreceptors, and physiological connections between these neurons and their clock neuron targets ensure light entrainment of the CCNN as a whole (Schlichting, 2016).

In both mammals and insects, the CCNN receives light input from multiple photoreceptor types. In Drosophila, the CCNN is entrained by photoreceptors in the compound eye, the ocelli, the Hofbauer-Buchner (HB) eyelets, and by subsets of clock neurons that express the blue light photoreceptor Cryptochrome (CRY). Understanding how multiple light input pathways modulate the CCNN to ensure entrainment to the environmental LD cycle is critical for understanding of the circadian system and its dysfunction when exposed to the unnatural light regimens accompanying much of modern life (Schlichting, 2016).

This study investigates the physiological basis and circadian role of a long-suspected circadian light input pathway in Drosophila: the HB eyelets. These simple accessory eyes contain four photoreceptors located at the posterior edges of the compound eyes and project directly to the accessory medullae (AMe), neuropils that support circadian timekeeping in insects. In Drosophila, the AMe contain projections from ventral lateral neurons (LNvs), important components of the CCNN that express the neuropeptide pigment dispersing factor (PDF), an output required for robust circadian rhythms in locomotor activity. The axon terminals of the HB eyelets terminate near PDF-positive LNv projections and analysis of visual system and cry mutants reveals a role for the HB eyelet in the entrainment of locomotor rhythms to LD cycles, but how the eyelets influence the CCNN to support light entrainment is not well understood (Schlichting, 2016).

This study presents evidence that this circadian light input pathway excites the small LNvs (s-LNvs) and acts to phase-dependently advance free-running rhythms in sleep/activity while inhibiting the large LNvs (l-LNvs). This work reveals that input from external photoreceptors differentially affects specific centers within the fly CCNN. Furthermore, it was shown that, under long summer-like days, the l-LNvs act to modulate subsets of so-called evening cells to delay the onset of evening activity. These results reveal a neural network underlying the photoperiodic adjustment of sleep and activity (Schlichting, 2016).

The experiments described in this study lead to two unexpected findings regarding the network properties of circadian entrainment in Drosophila. First, the l-LNvs govern the phase of evening peak of activity through PdfR-dependent effects on evening cells that bypass the s-LNvs. Although previous work has implicated the l-LNvs in the control of evening peak phase, the current results are the first to provide evidence that there is a direct connection between the l-LNvs and evening cells within the AMe and that this connection mediates the photoperiodic adjustment of sleep and activity in the fly. Second, the HB eyelets light input pathways, long implicated in circadian entrainment, have opposing effects on the l-LNvs and s-LNvs, inhibiting the former and exciting the latter. These results reveal not only a differential effect of a light input pathway on specific nodes of the CCNN but also establish that light from extraretinal photoreceptors can have synergistic or antagonistic effects on CRY- and compound eye-mediated light responses, depending on the clock neuron target in question (Schlichting, 2016).

Both the l-LNvs and s-LNvs express the blue light circadian photoreceptor CRY, the expression of which renders neurons directly excitable by light entering the brain through the cuticle. How such CRY-mediated light input interacts with input from external photoreceptors is not well understood, although it is known that each system alone is sufficient for the entrainment of locomotor rhythms. Genetic evidence suggests that the HB eyelets have relatively weak effects on circadian entrainment: flies with functional eyelets that lack compound eyes, ocelli, and CRY entrain relatively poorly to LD cycles relative to flies with functional eyes or CRY. The small phase responses of locomotor rhythms to HB eyelet excitation further supports a relatively weak effect of the eyelet on free-running locomotor rhythms (Schlichting, 2016).

The LNvs are critical nodes in the CCNN and are closely associated with input pathways linking the central brain to external photoreceptors. Work on the LNvs has provided evidence for a division of labor among the l-LNvs and s-LNvs: the l-LNvs are wake-promoting neurons that acutely govern arousal and sleep independently of the s-LNvs, whereas the s-LNvs act as key coordinators of the CCNN to support robust circadian timekeeping. Anatomical and genetic evidence has long supported the notion that the dorsal projections of the s-LNvs represent the key connection between the LNvs and the remaining components of the CCNN. However, a smaller body of work has suggested that the l-LNvs also contribute to the entrainment of sleep/activity rhythms under LD cycles. The Pdf knockdown and PdfR rescue experiments under long day conditions indicate that, as the day grows longer, the l-LNvs play a greater role in the timing of the evening peak. Moreover, the effects of PDF released from the l-LNvs are mediated not by the PDF receptive s-LNvs but rather by the fifth s-LNvs and a subset of the LNds, the NPF and ITP coexpressing LNds in particular (with some influence of the other PDF-receptor positive LNds). These same neurons were recently identified as evening cells that are physiologically responsive to PDF but relatively weakly coupled to LNv clocks under conditions of constant darkness. The results suggest that the l-LNvs differentially modulate the NPF/ITP-positive evening oscillators as a function of day length, producing stronger PDF-dependent delays under long day conditions through increased release of PDF from the l-LNvs, thereby delaying the evening activity peak. Thus, the l-LNvs mediate their effects on the evening peak of activity through their action on the NPF/ITP-positive subset of evening oscillators. The proposed PDF release from the l-LNvs under long days requires their activation via CRY and/or the compound eyes via ACh release from lamina L2 interneurons. It is hypothesized that the inhibitory influence of the HB eyelets ceases under long days allowing the compound eyes and CRY to maximally excite the l-LNvs. Indeed, previous work has established that the compound eyes are especially important for adapting fly evening activity to long days. Furthermore, several studies have suggested that the compound eyes signal to the l-LNvs leading to enhanced PDF release and a slowing-down of the evening oscillators. A recent paper measuring Ca2+ rhythms in the different clock neurons in vivo supports this view (Liang, 2016): Ca2+ rhythms in the l-LNvs peak in the middle of the day, unlike the s-LNvs, which display Ca2+ peaks in the late night/early morning. It is suggested that this phasing is produced by the inhibition of l-LNvs by the eyelets in the morning, followed by the excitation of the l-LNvs by the compound eyes and CRY. Interestingly, the only other clock neuron classes to display Ca2+ increases during the day are the LNds and fifth-sLNv, which phase lag the l-LNvs by ~2.5 h and display peak Ca2+ levels in the late afternoon, a time that coincides with the evening peak of activity (Liang, 2016). It is proposed that the relative coordination of Ca2+ rhythms between the l-LNvs and the LNds/fifth-sLNv is produced by the connection this study has identified between these neurons and the action of the eyelet and visual system on the l-LNvs (Schlichting, 2016).

Recent work has revealed that evening activity is promoted directly by the evening oscillator neurons and that the mid-day siesta is produced by the daily inhibition of evening oscillators by a group of dorsal clock neurons (Guo, 2016). It is proposed that the connections described in this study govern the timing of the evening peak of activity through the PDF-dependent modulation of the molecular clocks within the evening oscillator neurons, although PDF modulation likely results in the excitation of target neurons, which would promote evening activity. The results reveal new and unexpected network properties underlying the entrainment of the circadian clock neuron network to LD cycles. Excitatory effects of light on the LNvs are differentially modulated by the HB eyelets via cholinergic excitation of the s-LNvs and histaminergic inhibition of the l-LNvs. The work further reveals PDF-dependent modulatory connections in the AMe between the l-LNvs and the s-LNvs and, most surprisingly, between the l-LNvs and a small subset of evening oscillators. This work indicates that the latter connection is critical for the adjustment of evening activity phase during long, summer-like days. This network model of entrainment reveals not only how CRY and external photoreceptors interact within specific nodes of the CCNN, but also how photoreception is likely to drive changes in CCNN output in the face of changing day length (Schlichting, 2016).

In order to provide organisms a fitness advantage, circadian clocks have to react appropriately to changes in their environment. High light intensities (HI) play an essential role in the adaptation to hot summer days, which especially endanger insects of desiccation or prey visibility. This study shows that solely increasing light intensity leads to an increased midday siesta in Drosophila behavior. Interestingly, this change is independent of the fly's circadian photoreceptor cryptochrome (CRY), and solely caused by a small visual organ, the Hofbauer-Buchner (HB) eyelets. Using receptor knockdowns, immunostaining, as well as recently developed calcium tools, the eyelets were shown to activate key core clock neurons, namely the s-LNvs, at HI. This activation delays the decrease of PER in the middle of the day and propagates to downstream target clock neurons that prolong the siesta. Together a new pathway is shown for integrating light intensity information into the clock network, suggesting new network properties and surprising parallels between Drosophila and the mammalian system (Schlichting, 2019).

The fruit fly Drosophila melanogaster has two types of external visual organs, a pair of compound eyes and a group of three ocelli. At the time of neurogenesis, the proneural transcription factor Atonal mediates the transition from progenitor cells to differentiating photoreceptor neurons in both organs. In the developing compound eye, atonal (ato) expression is directly induced by transcriptional regulators that confer retinal identity, the Retinal Determination (RD) factors. Little is known, however, about control of ato transcription in the ocelli. Here we show that a 2kb genomic DNA fragment contains distinct and common regulatory elements necessary for ato induction in compound eyes and ocelli. The three binding sites that mediate direct regulation by the RD factors Sine oculis and Eyeless in the compound eye are also required in the ocelli. However, in the latter, these sites mediate control by Sine oculis and the other Pax6 factor of Drosophila, Twin of eyeless, which can bind the Pax6 sites in vitro. Moreover, the three sites are differentially utilized in the ocelli: all three are similarly essential for atonal induction in the posterior ocelli, but show considerable redundancy in the anterior ocellus. Strikingly, this difference parallels the distinct control of ato transcription in the posterior and anterior progenitors of the developing compound eyes. From a comparative perspective, these findings suggest that the ocelli of arthropods may have originated through spatial partitioning from the dorsal edge of an ancestral compound eye (Zhou, 2016).